Method for preparing BVDV-1 vaccine by non-cap-dependent mRNA (messenger ribonucleic acid) technology

Abstract

The invention relates to the technical field of vaccine preparation, and discloses a method for preparing a BVDV-1 vaccine by a non-cap-dependent mRNA (messenger ribonucleic acid) technology, which is characterized in that an IRES sequence of CVB3 is inserted into the upstream of a BVDV-1E2 gene to replace a 5'end cap structure of the traditional mRNA, so that the production process is obviously simplified, the dependence on expensive enzymatic reaction is reduced, the vaccine production cost is reduced, and the production efficiency is improved. The method is especially suitable for large-scale economic sensitive scenes such as animal husbandry. According to the present invention, the non-capped mRNA is encapsulated by using the lipid nanoparticle (LNP) with the specific molar ratio, such that the stability of the mRNA and the target cell delivery efficiency are effectively improved, the immunogenicity limitation caused by insufficient translation efficiency of the non-capped mRNA is overcome, and the immune response equivalent to the commercial vaccine is achieved in the large animal. The safety advantage is particularly prominent, inflammatory response or abnormal hemogram is not observed in all animal models, a novel solution with high efficiency, safety and economical efficiency is provided for prevention and control of livestock epidemic diseases, and the method is particularly suitable for animal infectious diseases such as bovine viral diarrhea needing long-term and extensive immunization.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and specifically to a method for preparing a BVDV-1 vaccine by a cap-independent mRNA technology. Background Art

[0002] Traditional mRNA vaccines rely on the 5′-end cap structure (Cap) to regulate translation efficiency and stability, and their preparation requires the addition of a Cap1 structure through enzymatic reactions (such as vaccinia capping enzyme and methyltransferase). This process significantly increases production costs and process complexity, especially facing problems such as enzyme activity dependence and cumbersome steps in large-scale production. Although capping mRNA vaccines exhibit excellent immunogenicity, their high production costs and patent barriers limit their application in economically sensitive fields such as animal husbandry.

[0003] In the prior art, cap-independent mRNA vaccines drive translation through an internal ribosome entry site (IRES). Although this can simplify the production process, it has the defect of insufficient immunogenicity. Research shows that the translation efficiency mediated by IRES is relatively low, resulting in significantly lower neutralizing antibody levels induced by it in small animal models compared to capping vaccines, making it difficult to meet actual immunization needs. In addition, the insufficient adaptability of the delivery system further limits the stability and cellular uptake efficiency of cap-independent mRNA. There is an urgent need for an optimized solution that takes into account cost, safety, and immunogenicity. For this reason, a method for preparing a BVDV-1 vaccine by a cap-independent mRNA technology is proposed. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a method for preparing a BVDV-1 vaccine by a cap-independent mRNA technology to solve the problems in the background art.

[0005] To achieve the above object, the present invention provides the following technical solution: A method for preparing a BVDV-1 vaccine by a cap-independent mRNA technology, the method comprising the following steps:

[0006] Construct a DNA plasmid containing the coding gene of the E2 protein of the BVDV-1 virus by molecular technology, and insert the internal ribosome entry site (IRES) of coxsackievirus B3 (CVB3) upstream of the gene;

[0007] Linearize the DNA plasmid by restriction enzyme digestion to ensure the integrity of the transcription template;

[0008] Perform in vitro transcription using T7 RNA polymerase to generate cap-independent mRNA;

[0009] After transcription is completed, remove the residual DNA template by DNase treatment;

[0010] Encapsulate uncapped mRNA in lipid nanoparticles (LNPs), where the LNPs are composed of DHA-1, DSPC, cholesterol, and DMG-PEG2000.

[0011] Preferably, the molar ratio of DHA-1, DSPC, cholesterol, and DMG-PEG2000 is 50:10:38:1.5.

[0012] Preferably, before encapsulating the uncapped mRNA, it also includes determining the purity and quality of the RNA using spectrophotometric analysis and agarose gel electrophoresis.

[0013] Preferably, the restriction enzyme digestion is BspQI, the plasmid design contains a 3′ polyadenylation sequence, and the restriction enzyme cleavage site is located after the 3′ polyadenylation sequence.

[0014] Preferably, the in vitro transcription temperature is 37 °C, and the transcription time is 2 hours.

[0015] Preferably, the temperature for incubating the DNase with the uncapped mRNA is 37 °C, and the incubation time is 15 minutes.

[0016] Preferably, the molecular technique specifically uses molecular cloning technology.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention inserts the IRES sequence of CVB3 upstream of the BVDV-1E2 gene, replacing the 5′-end cap structure of traditional mRNA, significantly simplifying the production process and reducing the dependence on expensive enzymatic reactions, thereby reducing the production cost of the vaccine and being particularly suitable for large-scale and cost-sensitive scenarios such as the livestock industry. By encapsulating uncapped mRNA with lipid nanoparticles (LNPs) at a specific molar ratio, the stability of the mRNA and the efficiency of target cell delivery are effectively improved, overcoming the immunogenic limitations caused by insufficient translation efficiency of non-cap-dependent mRNA, and achieving an immune response equivalent to that of commercial vaccines in large animals. Its safety advantages are particularly prominent, and no inflammatory reactions or abnormal blood profiles are observed in all animal models, providing a new solution with high efficiency, safety, and economy for the prevention and control of livestock diseases, especially for animal infectious diseases such as bovine viral diarrhea that require long-term and extensive immunization.

[0019] Other features and advantages of the present invention will be described in the following specification, and part of them will become obvious from the specification or be understood by implementing the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the structures pointed out in the specification, claims, and drawings. Brief Description of the Drawings

[0020] Figure 1Schematic diagram of the design of the cap-dependent (A) and cap-independent (B) mRNA vaccines of the present invention;

[0021] Figure 2 Schematic diagram of the efficacy of the mRNA vaccine of the present invention in a mouse model;

[0022] Figure 3 Schematic diagram of the efficacy of the mRNA vaccine of the present invention in guinea pigs;

[0023] Figure 4 Table showing the body weights of mice of the present invention;

[0024] Figure 5 Table showing the changes in neutralizing antibody titers in mouse sera of the present invention;

[0025] Figure 6 Table showing the differential white blood cell counts of mice on the 56th day of the present invention;

[0026] Figure 7 Table showing the body weights of guinea pigs of the present invention;

[0027] Figure 8 Table showing the titers of neutralizing antibodies in guinea pig sera of the present invention;

[0028] Figure 9 Table showing the differential white blood cell counts of guinea pigs on the 56th day of the present invention;

[0029] Figure 10 Table showing the body weights of goats of the present invention;

[0030] Figure 11 Table showing the rectal temperatures of goats of the present invention;

[0031] Figure 12 Table showing the hemogram analysis of goats of the present invention;

[0032] Figure 13 Table showing the titers of neutralizing antibodies in goat sera of the present invention. Detailed implementation manners

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art in the technical field based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0034] Please refer to the figure. A method for preparing a BVDV-1 vaccine using the non-capped mRNA technology in the present invention drives translation through an internal ribosome entry site (IRES), replaces the traditional mRNA capping mechanism, and combines a specific lipid nanoparticle (LNP) delivery system to achieve a low-cost and high-safety livestock vaccine.

[0035] 1. DNA plasmid construction

[0036] a. Gene design:

[0037] Select the E2 envelope protein gene of BVDV-1 as the target antigen, and insert the IRES sequence of coxsackievirus B3 (CVB3) upstream of its start codon to drive non-capped translation.

[0038] Insert the above sequence into the pUC57 plasmid vector through molecular cloning technology to form a recombinant DNA plasmid.

[0039] b. Plasmid linearization:

[0040] Use the restriction endonuclease BspQI to digest the plasmid. The digestion site is located after the 3′ polyadenylate (poly-A) sequence to ensure the integrity of the transcription template.

[0041] Verify the digestion product by agarose gel electrophoresis and purify the linearized DNA using a gel extraction kit.

[0042] 2. In vitro transcription of non-capped mRNA

[0043] a. Transcription reaction:

[0044] Use the T7 High Yield RNA Transcription Kit (Vazyme TR101) to perform in vitro transcription of the linearized DNA at 37°C for 2 hours to generate non-capped mRNA.

[0045] Reaction system: 1 μg of linearized DNA template, 2 μL of T7 RNA polymerase, 8 μL of NTP mixture (25 mM), 10 μL of reaction buffer, add nuclease-free water to 50 μL.

[0046] b. Purification and quality control:

[0047] After the transcription is completed, immediately add DNase I (1 U / μL) and incubate at 37°C for 15 minutes to degrade the residual DNA template.

[0048] Determine the concentration (A260 / A280 ratio ≥ 2.0) by spectrophotometric analysis (NanoDrop One / OneC) and verify the integrity by agarose gel electrophoresis (clear band, no degradation).

[0049] 3. Lipid nanoparticle (LNP) encapsulation

[0050] a. LNP formulation:

[0051] DHA-1, DSPC, cholesterol, and DMG-PEG2000 were weighed at a molar ratio of 50:10:38:1.5 and dissolved in ethanol to form a lipid mixture.

[0052] The uncapped mRNA (concentration 100 ng / μL) was dissolved in citrate buffer (pH 4.0), and the lipid mixture was mixed with the mRNA solution at a volume ratio of 3:1 by microfluidics technology to self-assemble into LNP.

[0053] b. LNP characterization:

[0054] The particle size was measured to be 90 ± 10 nm using dynamic light scattering (DLS), the polydispersity index (PDI) < 0.2, and the encapsulation efficiency ≥ 90%.

[0055] 4. Animal immunization experiments

[0056] Immunization experiments were conducted in mice, guinea pigs, and goats. Mice and guinea pigs were inoculated with 10 μg and 20 μg of uncapped mRNA vaccine respectively, and goats were inoculated with 25 μg of uncapped mRNA vaccine. After immunization, the body weight, white blood cell count, and neutralizing antibody titer of the animals were monitored.

[0057] 1. Mouse experiment

[0058] Thirty 6-8-week-old female BALB / c mice were divided into two groups and inoculated with 10 μg of uncapped mRNA vaccine respectively. Immunization was carried out on day 0 and day 21, with alternate injection into the left and right hind limbs. After immunization, the body weight, white blood cell count, and neutralizing antibody titer of the mice were monitored. As Figure 2 shown, where: (A) represents the timeline of the mouse experiment design; (B) represents the normalized body weight of the immunized mice (n = 15 per group). The red arrows indicate vaccination (first dose) and boost (second dose). (C) represents the neutralizing antibody titers against BVDV-1 on day 0, day 35, day 42, day 49, and day 56 according to immunofluorescence inhibition (n = 3 per group, and each serum sample was pooled from 5 mice). (D) represents the effect of vaccination on white blood cells (WBC), lymphocytes (LYM), intermediate cells (IMD), and granulocytes (GRA) on day 56.

[0059] 2. Guinea pig experiment

[0060] Six 6-8-week-old female guinea pigs were divided into two groups and inoculated with 20 μg of uncapped mRNA vaccine respectively. Immunization was carried out on day 0 and day 21 by intramuscular injection. After immunization, the body weight, white blood cell count, and neutralizing antibody titer of the guinea pigs were monitored.

[0061] 3. Goat Experiment

[0062] Nine 3 - 4 - year - old female goats were divided into three groups and inoculated with 25 μg of the uncapped mRNA vaccine. Immunization was carried out on day 0 and day 21 by intramuscular injection into the neck muscles. After immunization, the body weight, rectal temperature, white blood cell count, and neutralizing antibody titer of the goats were monitored. As Figure 3 shown, where: (A) represents the experimental design diagram of guinea pigs. (B) represents the normalized body weight of immunized guinea pigs (n = 3 per group). The red arrows indicate the primary immunization and booster immunization. (C) represents the BVDV - 1 - specific neutralizing antibody titers in serum detected by immunofluorescence inhibition on day 0, day 28, day 35, day 42, day 49, and day 56 (n = 3 per group). (D) represents the effects of vaccination on white blood cells (WBC), lymphocytes (LYM), intermediate cells (IMD), and granulocytes (GRA) on day 56 (n = 3 per group, represented by black dots).

[0063] 4. Experimental Results

[0064] 1. Efficacy of BVDV - 1 mRNA Vaccine in Mice

[0065] The neutralizing antibody titer induced by the uncapped mRNA vaccine in mice reached 7.9 (expressed as - log2) on day 35, which was significantly lower than that of the capped mRNA vaccine. The body weight of the mice remained stable after immunization, and no obvious side effects were observed. The results of differential white blood cell count showed that the white blood cells, lymphocytes, intermediate cells, and granulocytes of all mice were within the normal range, as Figures 4 - 6 shown.

[0066] 2. Efficacy of BVDV - 1 mRNA Vaccine in Guinea Pigs

[0067] The neutralizing antibody titer induced by the uncapped mRNA vaccine in guinea pigs reached 9.1 (expressed as - log2) on day 35, which was significantly lower than that of the capped mRNA vaccine. The body weight of the guinea pigs remained stable after immunization, and no obvious side effects were observed. The results of differential white blood cell count showed that the white blood cells, lymphocytes, intermediate cells, and granulocytes of all guinea pigs were within the normal range, as Figures 7 - 9 shown.

[0068] 3. Efficacy of BVDV - 1 mRNA Vaccine in Goats

[0069] The neutralizing antibody titer induced by the capped mRNA vaccine in goats was 10.2, showing no significant difference compared with the commercial vaccine. The body weight, rectal temperature, and white blood cell count of the goats remained normal, and no obvious side effects were observed, as Figures 10 - 13 shown.

[0070] In this example, a cap-independent mRNA vaccine containing the BVDV-1 E2 gene and the CVB3 IRES was constructed and combined with lipid nanoparticles to verify its safety and immunogenicity in mice, guinea pigs, and goats: The neutralizing antibody titers induced by the uncapped vaccine in small animals were significantly lower than those of the capped vaccine, but in goats, it was comparable to the commercial vaccine, and all safety indicators such as the body weight and blood picture of the animals were normal. The results showed that this technology replaced the traditional capping mechanism with the IRES, simplified the production process, and provided a new low-cost and safe strategy for the development of livestock vaccines.

Claims

1. A method for preparing a BVDV-1 vaccine using cap-independent mRNA technology, characterized in that: The method comprises the following steps: A DNA plasmid containing a gene encoding the E2 protein of BVDV-1 virus was constructed by molecular techniques, wherein the internal ribosome entry site (IRES) of Coxsackievirus B3 (CVB3) was inserted upstream of the gene; Linearize the DNA plasmid by restriction enzyme digestion to ensure the integrity of the transcription template; In vitro transcription was performed using T7 RNA polymerase to generate non-capped mRNA; After transcription is completed, residual DNA template is removed by DNase treatment; The non-capped mRNA was encapsulated in lipid nanoparticles (LNPs) composed of DHA-1, DSPC, cholesterol and DMG-PEG2000.

2. The method for preparing a BVDV-1 vaccine using cap-independent mRNA technology according to claim 1, characterized in that: The molar ratio of DHA-1, DSPC, cholesterol and DMG-PEG2000 is 50:10:38:1.

5.

3. The method for preparing a BVDV-1 vaccine using cap-independent mRNA technology according to claim 1, characterized in that: Prior to uncapped mRNA encapsulation, the purity and quality of the RNA were determined using spectrophotometric analysis and agarose gel electrophoresis.

4. The method for preparing a BVDV-1 vaccine using cap-independent mRNA technology according to claim 1, characterized in that: The restriction enzyme is BspQI, the plasmid design contains a 3' polyadenylic acid sequence, and the restriction enzyme site is located after the 3' polyadenylic acid sequence.

5. The method for preparing a BVDV-1 vaccine using cap-independent mRNA technology according to claim 1, characterized in that: The in vitro transcription temperature was 37° C., and the transcription time was 2 hours.

6. The method for preparing a BVDV-1 vaccine using cap-independent mRNA technology according to claim 1, characterized in that: The DNase was incubated with non-capped mRNA at 37° C. for 15 minutes.

7. The method for preparing a BVDV-1 vaccine using cap-independent mRNA technology according to claim 1, characterized in that: The molecular technology specifically adopts molecular cloning technology.

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